Local environment in biomolecular condensates modulates enzymatic activity across length scales

The mechanisms that underlie the regulation of enzymatic reactions by biomolecular condensates and how they scale with compartment size remain poorly understood. Here we use intrinsically disordered domains as building blocks to generate programmable enzymatic condensates of NADH-oxidase (NOX) with different sizes spanning from nanometers to microns. These disordered domains, derived from three distinct RNA-binding proteins, each possessing different net charge, result in the formation of condensates characterized by a comparable high local concentration of the enzyme yet within distinct environments. We show that only condensates with the highest recruitment of substrate and cofactor exhibit an increase in enzymatic activity. Notably, we observe an enhancement in enzymatic rate across a wide range of condensate sizes, from nanometers to microns, indicating that emergent properties of condensates can arise within assemblies as small as nanometers. Furthermore, we show a larger rate enhancement in smaller condensates. Our findings demonstrate the ability of condensates to modulate enzymatic reactions by creating distinct effective solvent environments compared to the surrounding solution, with implications for the design of protein-based heterogeneous biocatalysts.


Results
• Figure 1: o E) It's not immediately clear that the FAD cofactor colocalizes with the NADH, and it's also not clear where the condensates are.The green FAD dots don't really line up with the blue NADH blobs, so we don't know which ones are in the same condensate and where the condensates are.A composite image which shows recruitment and localizafion of NADH and FAD to brighffield condensates would be very helpful.
• Line 167-175: It would be helpful to provide more explanafion as to what exactly you're measuringhow much substrate and cofactor is in the condensate compared to the dilute phase?How did you calculate this?Even if the explanafion is in methods, it would be nice to have a brief descripfion here.In addifion, there should be a sentence describing what the determined parfifion coefficients actually show -increased posifive charge befter recruits NADH and FAD?
• Figure 3: o C) is unclear.If the protein concentrafion was close to 300nM, but not at300nM, why does the schemafic show protein concentrafion at 300nM?What protein concentrafion was actually used?That should be market on the graph.Authors should also have some other values on the X axis to give a sense of scale of the binodal.Furthermore, it's unclear what the Y axis represents.
• Line 264-265: Previously, authors said protein concentrafions were "close to" 300nM.Here, the authors say the solufions were 300nM.If the saturafion concentrafion is 300nM, wouldn't the proteins undergro phase transifion and form condensates?The authors should be more precise in these paragraphs.
Finally, a recommended experiment-augtorscan incubate cofactors with only LCD condensates first (concentrafion required may be different than that for fusions), followed by addifion of free NOX .If LCDs are sequestering the substrates (as author's suggest it recruitment of substrates and not NOX), the enzyme acfivity trend should reverse.

Reviewer #2 (Remarks to the Author):
Gil-Garcia et al. present an interesfing work on enzymafic catalysis of NADH oxidase (NOX) in confined microenvironments formed by designed protein condensates.Rather than encapsulafing the enzyme NOX in the condensates, as many other have shown before, the authors conjugated NOX to low complexity domains (LCDs) from three difference intrinsically disordered proteins, and thus the condensate building block is also the catalyst.This unique approach was previously reported by the authors.In this study, the authors focus on how the chemical composifion, and specifically the charge of three different LCDs affect the kinefics of NOX.By using SEC, DLS, and different spectroscopy and microscopy techniques, the authors analyzed the recruitment of conjugated NOX, substrate and cofactor in the condensates and the reacfion kinefics in the dilute phase and overall averaged heterogenous condensate solufions.The most interesfing observafion of this work is that condensate size does not affect Kcat but rather the basic microenvironment created by LLPS of Dpb 1.The mechanism by which the basic environment enhances enzyme catalysis remains unknown, although the authors suggested a few possible explanafions.Overall, the paper reads well and presents important insights on how compartmentalizafion (specifically size and charge of compartments) affects enzymafic catalysis.The thorough analyses and solid data overall support the conclusions of the work, yet I encourage the authors to consider my suggesfions as detailed below.I believe that the insights from the work will be of great interest to the growing LLPS community and to the broader readership of Nat.Commun., therefore I recommend on publicafion of the work after addressing the following comments: 1. Characterizafion of LLPS: There is liftle informafion on the formafion of condensates -the authors menfion later in the text that the Csat is 300 nM but no phase diagrams are presented to support this.What triggers LLPS? is it salt (20 mM NaCl)?Specifically, the authors stated that "electrostafic interacfions are important in the condensafion of the selected LCDs", I'm assuming that the Dbp-NOX will require higher salt concentrafion to trigger LLPS due to stronger electrostafic repulsion?It will be useful to elaborate on this.
2. Figure 1: (i) Were the microscopy images in panel c of figure 1 taken using phase contrast microscope?If so, please indicate.(ii) Why did the authors use 5 µM of conjugated enzyme for the microscopy analysis and a different concentrafion (1 µM) to analyze the reacfion kinefics?3. Follow up quesfion: how was the concentrafion of conjugated enzyme selected?please indicate in Figure 2 capfion the protein concentrafion and condifions used in the reacfion kinefics analysis.

Page 6 line 149:
The line reads as the authors analyzed the recruitment of free NOX rather than the full-length building block (NOX conjugated to LCDs), please rephrase to clarify this point.

5.
Have the authors considered analyzing substrate and cofactor parfifioning using a complementary method in a similar manner to the enzyme recruitment analysis by using averaged absorbance/fluorescence spectroscopy?This can provide useful informafion and validafion of the confocal analysis.
6.I am curious if the material properfies of the difference condensates are different due to their varying net charge.Have the authors analyzed the dynamics of the condensates using FRAP?Is it possible that the diffusion in the condensates affect reacfion rate?This is not mandatory but can add some valuable informafion to the work.
7. The rate of the enzymafic reacfion is measured by the decrease in absorbance of the NADH substrate.Have the authors considered the possible contribufion of light scaftering by the condensates, which might change over fime due to droplet coalescence?8. Do the authors expect that NOX forms dimers also in the dense phase?Does the enzyme form dimers or other type of oligomers in its nafive form?What is the enzyme pI?Please add this informafion to the MS.Also, is the enzymafic acfivity (of free NOX) not compromised by 0.5 M NaCl?Informafion about the enzyme (pI) might also help explaining the possible interacfions with the Dbp 1 LCD as suggested by the authors in the Discussion.9.The authors state that "for Dbp-NOX, the reacfion rate in the dilute phase was lower than the total reacfion rate of the two-phase system".Is it not obvious?How does the fact that the rate in the dilute phase is lower than the dense phase confirms the contribufion of the dense phase to the overall enzymafic reacfion, considering that the enzyme concentrafion in the dense phase is much higher?Please remove this statement of rephrase to clarify this point.
10. Are the 100 nm nanoclusters in the dilute phase detecfible using electron microscopy?This is not a mandatory analysis but can confirm the DLS results.
11. Figure 4c shows kinefics parameters of 10 nm sized clusters.What is the expected size of the building block (conjugated LCD-NOX)?Could the 10 nm peaks in DLS represent individual building blocks rather than structures?12. Discussion: "the increase in acfivity measured in Dbp 1 LCD condensates may indicate that the effect of substrate colocalizafion becomes important only above a certain threshold".Please explain this statement.

Reviewer #3 (Remarks to the Author):
The manuscript by Gil-Garcia et all describes a systemafic study of the effect of biomolecular condensates on enzymafic rates.This is an important topic that may help us understand both fundamental biological mechanisms and engineering new biocatalysts.The study compares three variants of the enzymes NADH oxidase fused to three different intrinsically disordered regions that drive phase separafion.The main finding is that one of these IDRs enhance the enzymafic reacfion about threefold, which the authors ascribe to an increase in the apparent kcat.This is in contrast to much of the literature on enzyme enhancement of condensates, which so far has mainly been documented to occur through mass acfion -i.e.co-parfifioning of enzyme and substrate.If the microenvironment truly enhances the fundamental rate constants this will be quite novel and to my mind surprising, but is not enfirely supported by the present manuscript.I think this calls for some extra analysis and possibly simple extra experiment to clarify whether the rate enhancement is due to up concentrafion of (co-)substrate or not.The manuscript itself also seems to be somewhat in doubt about this, as the enhancement is somefimes aftributed to kcat and somefimes to co-parfifioning.

Specific comments:
1.The key quesfion in my mind is to clarify whether the rate enhancement is due to substrate coparfifioning or not.Two out of the three substrates parfifion into the condensate with the enzyme.All other factors being equal, this suggests that the rate should be enhanced by mass acfion.When no enhancement is observed for two out of three this suggests a "cancellafion of effects", where rate enhancement due to mass acfion is cancelled by e.g.lower kcat or mass transport limitafions.A good starfing point would be to decompose the rates into the contribufion from the two phases theorefically.The authors do the inifial steps towards doing this by defining rates rI and rII on line 241.I think this analysis should be carried to the end to see what the actual rates are in the dense phase and compare them to predicfions from simple mass acfion.
2. An alternafive explanafion to the rate enhancement is the elevated concentrafion of the co-substrate in the condensate.To rule out this effect, it would be nice to see the dependence of the rates on the FAD concentrafion.3. Fig. 4 -Michaelis-Menten dependence -The Michaelis-Menten curve and the simple effect via the apparent kcat is surprising.A similar KM means that the true KM of the dense phase has to be 12.5-fold greater (as the concentrafion of NADH is this much greater.Again, not impossible but a remarkable coincidence that should be discussed.I also think the non-Michaelis-Menten behavior of the two other enzymes (see below) call for a wider range of concentrafions to be tested.4. L. 189 "showing similar relafive trends between the different systems" -Is this really true?Two of the enzymes show a decrease at high concentrafions of NADH, which suggests a non Michaelis-Menten mechanism.This is glossed over and somewhat mis-represented by the statement above.There are good reasons why the rate may drop, but it should be discussed openly and included in the main manuscript. 5. Oxygen is also a substrate and could in principle also contribute to the rate enhancement.It would be very difficult to measure parfifioning of oxygen, and small molecules do not have parficularly large parfifioning constants, so I think this is unlikely.However, it would perhaps be worth menfioning in passing, and for example state if it is saturated with oxygen under the condifions used.6.The kinefics of Dbp1-NOX in Fig 2A are clearly mulfi-phasic in contrast to the other proteins.This is the protein that the enfire story hinges on, so I think we need an explanafion for this.The inifial fast burst phase is really what drives the difference between studies.Is there something fundamentally different about this reacfion that make it mulfiphasic?
Minor issuses: 1. Could the sequences in the SI be added as text rather than images to make them machine-readable and easier to copy? 2. 2. L. 171 -You cannot conclude that they scale exponenfially from 3 data points.Answer from the authors: We thank the reviewer for the suggestion.We have removed the word "typically" and rephrased accordingly in the new version of the manuscript.
"Moreover, condensates in cells can not only populate the microscale, but also the nanoscale 30 "

Results
• Figure 1: o E) It's not immediately clear that the FAD cofactor colocalizes with the NADH, and it's also not clear where the condensates are.The green FAD dots don't really line up with the blue NADH blobs, so we don't know which ones are in the same condensate and where the condensates are.

Answer from the authors:
We appreciate the reviewer's observation.Due to the short timescale of the reaction, to evaluate the partition coefficient we had to conduct two distinct experiments, adding substrate and cofactor individually.This allows to avoid interference of the fast reaction while measuring the partitioning coefficient.We have now specified in the caption of Figure 1 that the experiments were conducted independently and further clarified this point in the main text (Results section "Condensate microreactors with different local environments").Results section: "To avoid any influence of the rapid reaction, we measured the partitioning of NADH and FAD independently by confocal microscopy.Specifically, we recorded their intrinsic fluorescence signal at 410-440 nm and 510-530 nm for NADH and FAD, respectively (Figure 1E), and calculated the partition coefficient (Kp) as the ratio of fluorescence intensity inside and outside the condensates." A composite image which shows recruitment and localization of NADH and FAD to brightfield condensates would be very helpful.
Answer from the authors: Prompted by the comment of the reviewer, we performed a co-localization experiment adding brightfield images, again by adding the two molecules individually in two different samples to avoid interference of the reaction (new Supplementary Figure 3).• Line 167-175: It would be helpful to provide more explanation as to what exactly you're measuringhow much substrate and cofactor is in the condensate compared to the dilute phase?How did you calculate this?Even if the explanation is in methods, it would be nice to have a brief description here.In addition, there should be a sentence describing what the determined partition coefficients actually showincreased positive charge better recruits NADH and FAD?

Answer from the authors:
We have now specified this information in the Results section "Condensate microreactors with different local environments" of the main text, and added the suggested sentence.
"Specifically, we recorded their intrinsic fluorescence signal at 410-440 nm and 510-530 nm for NADH and FAD, respectively (Figure 1E), and calculated the partition coefficient (Kp) as the ratio of fluorescence intensity inside and outside the condensates.
"Both molecules specifically partition into the condensates, as confirmed by the overlap between the fluorescence and bright-field images for Dbp1-NOX condensates (similar results were obtained for the other LCD-NOX constructs) (Suppl.Fig. 3).These findings confirm the increased uptake of the negatively-charged substrate and cofactor by the most cationic condensates." • Figure 3: o C) is unclear.If the protein concentration was close to 300nM, but not at 300nM, why does the schematic show protein concentration at 300nM?What protein concentration was actually used?That should be market on the graph.
Answer from the authors: The csat was estimated from the measurement of the concentration in the dilute phase after separation of the dense phase, and was equal to 290-300 nM (Table 1).This value has been now further confirmed by new experiments on phase diagrams performed to address point 1 of reviewer 2 (new Supplementary Figure 7 and Supplementary Figure 8).We performed experiments at 280 nM to be close to this value.At this protein concentration we see only a small amount of very tiny condensates.We have now specified that experiments were performed at 280 nM in both the figure and the text.
Authors should also have some other values on the X axis to give a sense of scale of the binodal.Furthermore, it's unclear what the Y axis represents.
We have added more values on the X axis as requested (see new Figure 3C below).Y axis represents temperature normalized by the interaction coefficient (χ).This has been now specified in the Figure caption.• Line 264-265: Previously, authors said protein concentrations were "close to" 300nM.Here, the authors say the solutions were 300nM.If the saturation concentration is 300nM, wouldn't the proteins undergro phase transition and form condensates?The authors should be more precise in these paragraphs.
Answer from the authors: Following the reviewer´s comment, we have now specified that the working concentration was 280 nM in the different sections and figures throughout the manuscript.
Finally, a recommended experiment-augtorscan incubate cofactors with only LCD condensates first (concentration required may be different than that for fusions), followed by addition of free NOX.If LCDs are sequestering the substrates (as author's suggest it recruitment of substrates and not NOX), the enzyme activity trend should reverse.
Answer from the authors: We thank the reviewer for this interesting suggestion.Unfortunately, the very low total volume fraction of the dense phase prevents this experiment, since an insufficient amount of substrate is depleted from the dilute phase to measure a detectable difference in the reaction in the dilute phase in this experiment.Specifically, considering the total volume fraction of 0.015%, even with a high partition coefficient of 50, less than 1% of the substrate would be sequestered into the LCD condensates.Moreover, we have observed that free NOX partially partitions into LCD condensates.

Reviewer #2 (Remarks to the Author):
Gil-Garcia et al. present an interesting work on enzymatic catalysis of NADH oxidase (NOX) in confined microenvironments formed by designed protein condensates.Rather than encapsulating the enzyme NOX in the condensates, as many other have shown before, the authors conjugated NOX to low complexity domains (LCDs) from three difference intrinsically disordered proteins, and thus the condensate building block is also the catalyst.This unique approach was previously reported by the authors.In this study, the authors focus on how the chemical composition, and specifically the charge of three different LCDs affect the kinetics of NOX.By using SEC, DLS, and different spectroscopy and microscopy techniques, the authors analyzed the recruitment of conjugated NOX, substrate and cofactor in the condensates and the reaction kinetics in the dilute phase and overall averaged heterogenous condensate solutions.The most interesting observation of this work is that condensate size does not affect Kcat but rather the basic microenvironment created by LLPS of Dpb 1.The mechanism by which the basic environment enhances enzyme catalysis remains unknown, although the authors suggested a few possible explanations.Overall, the paper reads well and presents important insights on how compartmentalization (specifically size and charge of compartments) affects enzymatic catalysis.The thorough analyses and solid data overall support the conclusions of the work, yet I encourage the authors to consider my suggestions as detailed below.I believe that the insights from the work will be of great interest to the growing LLPS community and to the broader readership of Nat.Commun., therefore I recommend on publication of the work after addressing the following comments: Answer from the authors: We thank the reviewer for the positive feedback and the constructive comments, which we have addressed as described in the point-to-point responses below.

Characterization of LLPS:
There is little information on the formation of condensatesthe authors mention later in the text that the Csat is 300 nM but no phase diagrams are presented to support this.
Answer from the authors: Thank you for pointing this out.We have rephrased this sentence in the new version of the manuscript.
"We next determined the recruitment of the different LCD-NOX fusion proteins into the different condensates by separating the dilute and dense phase by centrifugation and measuring the protein concentration in the dilute phase by size exclusion chromatography (SEC)." 5. Have the authors considered analyzing substrate and cofactor partitioning using a complementary method in a similar manner to the enzyme recruitment analysis by using averaged absorbance/fluorescence spectroscopy?This can provide useful information and validation of the confocal analysis.
Answer from the authors: We thank to the reviewer for this interesting suggestion.We tried is to separate dilute and dense phase and measure remaining concentration of substrate and cofactor in the dilute phase, comparing with a control homogeneous solution.However, this was not feasible due to the very low total volume of the dense phase and the minor changes in the concentration of the substate in the dilute phase upon recruitment in the dense phase.Specifically, if we consider the total volume fraction of 0.015%, even with a high partition coefficient of 50, less than 1% of the substrate amount would be sequestered into the condensates, making very difficult to observe any change in the absorbance/fluorescence of these molecules via spectroscopy.
6.I am curious if the material properties of the difference condensates are different due to their varying net charge.Have the authors analyzed the dynamics of the condensates using FRAP?Is it possible that the diffusion in the condensates affect reaction rate?This is not mandatory but can add some valuable information to the work.
Answer from the authors: The reviewer raised a very interesting point.However, this is a complex problem that requires a dedicated work and is out of the scope of this paper.
7. The rate of the enzymatic reaction is measured by the decrease in absorbance of the NADH substrate.Have the authors considered the possible contribution of light scattering by the condensates, which might change over time due to droplet coalescence?Answer from the authors: We appreciate the reviewer's comment.To address this concern, we performed a time course experiment in which we recorded Abs340nm of NADH in the presence of droplets of Dbp1-NOX and in the absence of any enzymatic reaction (we did not add the cofactor (FAD)).The goal was to determine whether, as suggested by the reviewer, the NADH absorbance values were affected by the condensate coalescence process over a 30-min period.This timescale is considerably longer than the time required for the enzymatic reaction (which occurs within minutes).As illustrated in the new Supplementary Figure 4, the absorbance values of NADH were unaffected by the light scattering of droplets.We specify this information in the revised version of the manuscript (Results section "Condensation with suitable local environment modulates NOX enzymatic activity").
"To confirm the absence of any potential effect of the condensates on the absorbance signal at 340 nm, we monitored the absorbance of NADH in the presence of Dbp1-NOX condensates in the absence of reaction (i.e., in the absence of FAD cofactor), observing a negligible change of the signal over time (Suppl.Fig. 4)." 3. Fig 1F -Error bars missingIntroduction• Line 76-78 -"condensates in cells are typically in the nanometer range".This claim may not be entirely correct, condensates can often reach micrometer size (e.g. the nucleolus, P-granules, etc).Ref:Forman-Kay et al., RNA 2022.

Figure : "
Figure: "Figure 1E: Representative fluorescence confocal microscopy images showing the recruitment of NADH (top, blue fluorescence) and FAD (bottom, green fluorescence) in condensates of Ddx4-NOX (left), Laf1-NOX (middle) and Dbp1-NOX (right).NADH and FAD were added individually in two distinct experiments to avoid interference of the rapid reaction.Scale bar represents 5 µm" The blue and green fluorescence signals of NADH and FAD overlap with the condensates observed in the bright-field images.Supplementary Figure 3. Substrate and cofactor partition into Dbp1-NOX condensates.Representative fluorescence and bright-field confocal microscopy images showing the recruitment of NADH alone (top, blue fluorescence) and FAD alone (bottom, green fluorescence) in Dbp1-NOX condensates.The merged images confirm the localization of substrate and cofactor within the condensates.

"Figure 3C :
Schematic phase diagram showing the protein concentration at which nanoclusters were formed (280 nM, red square), and the c sat (300 nM).The Y-axis represents temperature normalized by the interaction coefficient (χ)."